Muscle stiffness indicating mission crew health in space

Muscle function is compromised by gravitational unloading in space affecting overall musculoskeletal health. Astronauts perform daily exercise programmes to mitigate these effects but knowing which muscles to target would optimise effectiveness. Accurate inflight assessment to inform exercise programmes is critical due to lack of technologies suitable for spaceflight. Changes in mechanical properties indicate muscle health status and can be measured rapidly and non-invasively using novel technology. A hand-held MyotonPRO device enabled monitoring of muscle health for the first time in spaceflight (> 180 days). Greater/maintained stiffness indicated countermeasures were effective. Tissue stiffness was preserved in the majority of muscles (neck, shoulder, back, thigh) but Tibialis Anterior (foot lever muscle) stiffness decreased inflight vs. preflight (p < 0.0001; mean difference 149 N/m) in all 12 crewmembers. The calf muscles showed opposing effects, Gastrocnemius increasing in stiffness Soleus decreasing. Selective stiffness decrements indicate lack of preservation despite daily inflight countermeasures. This calls for more targeted exercises for lower leg muscles with vital roles as ankle joint stabilizers and in gait. Muscle stiffness is a digital biomarker for risk monitoring during future planetary explorations (Moon, Mars), for healthcare management in challenging environments or clinical disorders in people on Earth, to enable effective tailored exercise programmes.


Lower back (MP5)
There were no significant differences found on ANOVA for the Multifidus muscle at MP5 (F[2,116] = 2.87, p = 0.0609) (Fig. 2c).The muscle showed no changes from preflight to during the inflight period or from inflight to postflight (Fig. 2c).There were, however, some differences between time periods observed when mean differences were compared to MDC values (MDC 22.8 N/m 42 ; pre vs inflight 41.12 N/m > MDC; in vs postflight 37.24 N/m > MDC; pre to postflight 3.88 N/m < MDC;).

Plantar fascia (MP1)
The fascia on the sole of the foot (MP1) showed no statistically significant change in stiffness from preflight to inflight or in postflight recovery (F[2,117] = 2.927, p = 0.0575), with wide participant variability.Mean differences

Linear mixed model analysis inflight and posflight
Visualisation of the case-wise relationship between number of days and stiffness indicated a quadratic relationship for both inflight and postflight changes for some sites of the muscular system (Fig. 3).

Inflight changes in stiffness
A significant quadratic fixed-effect relationship was found between number of inflight days and stiffness for MP1 (p = 0.002), MP2 (p = 0.036), MP3 (p = 0.02) and MP9 (p = 0.04).The measurements sites related to tendons/ligaments (i.e.MP1, MP2, and MP9) all demonstrated a decrease in stiffness over time (Fig. 3a).The soleus muscle site (MP3) demonstrated an increase in stiffness over time (Fig. 3b).The remaining six sites (Supplementary Fig. S5) did not demonstrate a significant quadratic relationship during inflight conditions.Please see Supplementary Table S1 for data supporting the p-values.

Skin temperature from thermal imaging (FLIR camera system)
There were no significant changes in skin temperature over time at any of the MPs, indicating stable experimental conditions.Skin temperature measured during baseline data collection (BDC) sessions varied between MPs, with MP1 having the lowest skin temperature (29.0 °C) and MP6 the highest (35.4 °C) (Supplementary Table S2).All measurements of skin temperature lay within the physiological range 43 .

Subcutaneous tissue thickness from ultrasound imaging
There were no significant changes in subcutaneous tissue thickness documented over time for each of the 10 MPs.(Supplementary Figs.S2 and S3).

Discussion
The MYOTONES experiment was the first to collect data on muscle health (passive stiffness during full body unloading in microgravity) in astronauts during an entire space mission cycle involving inflight measurements at four time points on the ISS.The study protocol was designed for autonomous and compliant use by astronauts onboard the ISS as a pioneering inflight health monitoring tool for real-time muscle status and postflight rehabilitation assessment.The technology used in the present study has improved ability to examine distribution of muscle loss.By using passive muscle stiffness [N/m] recordings as digital biomarkers, we showed that most of the skeletal muscles and tendons studied are targeted effectively by inflight countermeasure exercise to mitigate microgravity-induced disuse, with the exception of Tibialis Anterior and the deep calf muscle Soleus.This evidence of the feasibility of selectively obtaining human muscle health data relatively simply within the unique space environment could pave the way for translation to enhanced global healthcare, through monitoring in everyday life, sports, aging populations and people living in challenging environments, as well as various clinical settings for musculoskeletal and neurological disorders on Earth.
The present novel insights indicate that a countermeasure exercise programme performed onboard the ISS generally preserves passive stiffness in muscle structures at most sites measured (shoulder, neck, back, thigh), as changes inflight showed only marginal differences from pre-and postflight measurements.Importantly, the Tibialis Anterior muscle (prime dorsiflexor, ankle joint stabilizer 44 , vital in human gait on Earth, showed reduced stiffness inflight in all 12 mission crew members studied in spite of routine inflight countermeasures currently available on the ISS 45 (T2, treadmill-2; ARED, advanced resistive exercise device; CEVIS, bicycle ergometer).This selective lack of preservation of stiffness, suggesting muscle loss/weakness, in an important muscle for gait is a new and unexpected finding, which requires consideration for ISS astronauts and longer-duration missions to Deep Space.
As part of the prime plantarflexor calf triceps surae (Soleus and Gastrocnemius) both antagonists to Tibialis Anterior, only Gastrocnemius (medial head) showed increased stiffness (Fig. 1c) inflight, in contrast to Soleus which showed a decrease (Fig. 1b).These inverse changes in stiffness may be due to the known muscle tissue diversity, i.e. fibre-type composition (Soleus predominantly slow fibers; Gastrocnemius mixed-fast-contracting fibres) 46 , and their functional characteristics 47 .Decreased stiffness in Soleus may be due to an altered force-length relationship (operating length) normally observed in human gait 27,48 which is likely compromised by gravitational unloading causing the plantarflexed ankle joint position of the relaxed astronaut´s body at rest (semi-squatting) in microgravity.This second novel observation from the space environment of inverse changes within the calf musculature has not been reported in previous space analogues, such as bed rest studies 49,50 but reiterates the need for standardized measurement conditions and appropriate body positioning during measurement, as these are key factors for reliable sampling and meaningful data interpretation 27 .Although Soleus muscle stiffness decreased compared to preflight, it did increase gradually over time, as demonstrated by a significant change in stiffness in a quadratic relationship with number of days spent on board the ISS.Therefore, it is possible that the countermeasures to mitigate the loss of muscle mass in microgravity help to preserve stiffness of the Soleus muscle to some extent.It is furthermore hypothesized that in space, Gastrocnemius at least partially overtakes function of the entire calf muscles (indicated by increased stiffness) with or without inflight countermeasures.Their mutual Achilles Tendon showed decreased stiffness inflight compared to preflight, which was also demonstrated in a quadratic relationship with number of days spent on board the ISS.The cause of decrease in stiffness is again likely due to the ankle joint of the fully unloaded body in microgravity being at a higher (plantaflexed) resting angle than on Earth.This results in less stretching and thus reduced storing and releasing elastic strain energy of the calf muscle-tendon unit in space than usually seen in terrestrial walking, running and jumping 51,52 on Earth as previously shown 53 .Monitoring Achilles tendon stiffness is critical for astronauts, as well as for healthy people of all ages on Earth 54 , since sudden reloading, such as gravitational force transitions and strenuous activity, can result in Achilles Tendon injury or even rupture 55 .Of particular note is that stiffness of the Achilles Tendon postflight was significantly lower than preflight and there was no significant relationship with stiffness and number of days post flight, in contrast to other tendons.Therefore, the risk of rupture for Astronauts might be high on strenuous activity, without appropriate reconditioning.The Achilles tendon rehabilitation process is relatively long and is known to have a high risk of tendon rerupturing 56 .Myoton technology used in the present study may prove to be an effective tool to monitor Astronauts' reconditioning after return from spaceflight but also the tendon's stiffness to evaluate healing processes and to adapt required therapeutic decisions on the magnitude of reloading of the foot in relevant clinical settings 57 .Reloading on return to gravity will also be vital to consider for partial gravity conditions during extravehicular activities in future planetary explorations (Moon/Mars) 4 .The exercise countermeasures inflight aim to minmise this risk of injury during realoading.
In contrast to the Achilles tendon, the Infrapatellar Tendon (tendinous link of Quadriceps Femoris muscle to the tibial bony tuberosity via the knee cap) showed increased stiffness in microgravity as the habitual lower limb semi-squatting posture in space is knee flexion, resulting in a stretched patellar tendon.Stiffness of Rectus Femoris (superficial mid-head of Quadriceps Femoris) remained unchanged in crew members onboard the ISS.The Rectus Femoris is a two-joint muscle (hip and knee) 58 and hip flexion, which is the main position adopted for the crew members' free-floating bodies in microgravity, probably counters the stretch effect of knee flexion.The high intensity inflight exercise usually performed by crew members on the advanced resistive exercise device (ARED) specifically targets the prime knee extensor, resulting in effectively maintaining Quadriceps muscle strength and stiffness 59 .However, it is important to note that there was a negative relationship in stiffness and number of days spent on the ISS for the Infrapatellar tendon inflight, so although stiffness increased pre-to inflight, it gradually declined inflight.As with the Achilles tendon, careful monitoring is important to track changes and identify potential risks for rupture or other injuries in the Space Medicine context.
Low back pain is a well-known problem following extended immobilization 60 and in astronauts in spaceflight 61 .Lumbar Multifidus atrophy and flattening of the lumbar lordosis have been reported in astronauts after long-duration spaceflight 62 .The higher stiffness of Multifidus observed inflight in the present study reduced significantly upon return, contrary to findings in a dry immersion study which reported reduced stiffness of the lower lumbar Erector spinae 63 , although a higher stiffness may be explained by inflight stretch of the muscle due to the flattening/elongation of the lower spine and a compression of the spine upon reloading 64  www.nature.com/scientificreports/pain is a major health issue on Earth due to high workload and seen in occupational medicine cohorts, such as nurses, delivery workers, office workers due to prolonged sitting, with less activity to maintain strength of the dorsal paravertebral muscle system and reduce strain on the spine itself 65 .Specifically, the Erector Spinae muscles extend and rotate the spine and Lumbar Multifidus stabilizes the spine (the latter monitored in the present study).Muscle stiffness detected by the Myoton technique has the potential to be a clinical indicator of spinal muscle health in order to quantitatively assess back pain syndromes in routine medical examination in real-time.
The variability in muscle stiffness between participants for gastrocnemius and rectus femoris was smaller than compared with other muscles and tendons (Figs. 1 and 2).This observation existed across the mission cycle, so was not related to effects of microgravity or countermeasures.Pre-flight data for Rectus Femoris (Fig. 2a), for example, were similar to mean and standard deviation values for 61 healthy young adults (males 292 ± 36; females 233 ± 35) reported by Agyapong-Badu et al. 29 .A possible technical reason for differences in variability may relate muscle size and the relative effects of force of the mechanical impulse from the Myoton device, which is standardized across muscles.The Rectus Femoris and Gastrocnemius are larger than most of the other muscles studied and inter-subject variability in body size would affect smaller muscles more than larger muscles.However, the Gastrocnemius and Soleus muscles are similar in size, yet their inter-subject variability differed (Fig. 1).Perhaps the homogeneity of muscles, e.g. in terms of fiber-type composition, is reflected in variability in their physiology and behavior.
Future long-duration missions into Deep Space will require compact design spacecraft with limited cabin space, providing less opportunity to perform countermeasure exercise using similar devices to those currently available on the ISS.Impaired muscle function increases the risk of poor performance during planetary excursions in partial gravity (e.g.Moon, Mars) but also increases the risk of falling upon return to Earth's gravity (1G).The variability of changes between astronauts observed in the present study suggests that personalized countermeasure exercises targeting specific muscle groups will be required during future long-duration missions to enable safer human planetary exploration 66 .Monitoring muscle health will be vital in this as yet unknown Deep Space environment and future countermeasure protocols need to be adjusted with respect to newly identified mission health risks of astronauts as shown in this work.In particular, close monitoring will be crucial for future Deep Space and Planetary Exploration where gait and postural stability are utmost requirements for human motion in altered gravity conditions (Moon, Mars), as well as for faster recovery on return to Earth.
Lessons from the present data regarding rapid, simple, non-invasive monitoring of muscle health have implications for assessing human health on Earth outside a laboratory environment, as this is not accessible for routine patient care.Such settings include: open field research; patients in remote situations, such as General Practitioner consultations, physiotherapy treatment sessions, hospital outpatient clinics and wards, and patients' homes.Critical care is a particularly relevant setting for continued monitoring, where patients' muscles can change rapidly on a daily basis 67 .Such monitoring (without the need for biopsy) would help more targeted treatments, as proposed in this work for astronauts on long-duration missions.Treatments and assessments of older people may also benefit from monitoring muscle health since sarcopenia (age-related muscle wasting) shows similar traits to muscle loss in microgravity 68 and stiffness changes with ageing have already been documented with Myoton technology 29 .
Lessons learned from spaceflight highlight some critical requirements for meaningful data acquisition from resting human muscle under normal 1G gravitational conditions on Earth.Deviation from the following recording conditions are likely cofounders of robust data acquisition using Myoton technology, some of which have been documented 27 : (i) fully relaxed body position (standing, sitting, lying); (ii) limb dominance (left/right); (iii) accurate location of skin measurement points (in the middle of a relaxed muscle belly/tendon length on superficial anatomical muscle/tendon units of interest); (iv) standardized joint positioning/angle (identical passive muscle lengthening) particularly when single-joint muscles (e.g.Soleus) versus two-/multiple-joint muscles (e.g., Biceps Brachii, Rectus Femoris, Gastrocnemius) as muscle of interest; (v) variable body composition/adiposity (excess of subcutaneous tissue/fat deposits over muscle of interest in obesity); (vi) avoidance of prior physical activity, which can increase muscle stiffness; (vii) and consistent time of day (diurnal variability) of measurements, as various aspects of muscle function vary, e.g.changes in resting muscle characteristics across the day were reported in professional athletes 69 .
Study limitations imposed by space-related research include: (i) lack of ability to perform electromyography (EMG signal silence to check for absence of electrophysiological input/muscle activity) during sessions inflight (operational constraint) 70 ; (ii) the relatively low number of astronauts (8 male vs. 4 female) prevented sound analysis of sex differences, as well as differences between novice astronauts and those who had flown previously (statistical constraint); (iii) slightly variable flight schedules and mission durations between astronauts prevented presentation of data at particular time points and, instead, mean values for time periods were used to enable comparisons across mission duration (statistical constraint).To account the limitation in this analysis, the separate linear mixed models analyses in Fig. 3 demonstrate changes over time both inflight and postflight; (iv) muscles examined was limited to 10 MPs due to time constraints but in principle, there is no limitation for the Myoton protocol to include many other superficial muscle groups of interest and relevance to more targeted exercise.Selection of the 10 MPs was based on relevance to movement/postural control on Earth, on a previous bed rest protocol 50 and knowledge of the behavior of various muscles in health and dysfunction.
The assessment protocol and definition of passive muscle stiffness as a muscle health indicator from the space environment could be helpful for many health professionals undertaking clinical assessments, and widespread uptake may result in a step-change for enhancing healthcare in neuro-musculoskeletal and geriatric medicine, rehabilitation and precision medicine on Earth.For example, assessment of stiffness and other muscle characteristics is very helpful in the management of neurological disorders such as Parkinson Disease (PD) 71 and stroke 38  www.nature.com/scientificreports/stiffness involves subjective protocols comprising manual palpation and evaluation of passive movements, and rating stiffness as mild, moderate or severe.As demonstrated in the astronaut study described, Myoton offers objective non-invasive measurement suitable for clinical environments to develop clinical scales, and for more accurate and sensitive evaluation of effects of treatments such as drugs 71 and brain stimulation as in PD 32 .Potential future applications also include home monitoring of drug effects, analogous to self-testing blood in diabetes.
The MYOTONES study brings a new dimension to translating the technology to clinical assessment through training novice users by remote guidance via teleconference.An example of such impact was realised with physiotherapists in Ghana who were guided remotely to establish reliability of using Myoton in PD patients 72 .
Areas where muscle strength cannot be measured due to: lack of equipment; pain from traumata/injuries, joint disorders (rheumatoid and osteoarthritis); during surgical interventions; or lack of cognition, such as in intensive care patients and in frail older people at risk of losing physical independence 29 ; are very promising examples of novel applications of the space-tested Myoton technology and protocol.Myoton also enables selective changes within a muscle group to be detected (e.g. the calf), which strength testing does not permit.
We conclude that stiffness was preserved in the majority of muscle structures studied but selective loss of stiffness of key muscles required for gait, despite daily inflight countermeasures, indicates more targeted exercises are required for effective countermeasures in space.Our work has contributed to the body of evidence for potential to translate Myoton technology to multiple other settings on Earth, from physical conditioning monitoring in remote areas or in extreme environments 73 to clinical rehabilitation enabling more targeted and efficiently tailored interventions.These applications based on muscle stiffness as digital biomarker open up new avenues for improved health status management in future Human Deep Space Exploration and for people on Earth.
Procedures involved non-invasive data collection with digitized Myoton technology on the 10 pre-selected individual skin measurement points (superficial muscle/fascia/tendon), ultrasound imaging by remotely controlled B-mode ECHO system provided by ESA (to measure subcutaneous tissue thickness at skin MPs to aid interpretation of Myoton recordings), and skin surface temperature detection via FLIR ThermoCam system (see below) at skin MPs (pre/postflight temperature range check; see Supplementary Table S1). .Due to contraints in crew time and to aid compliance, the inflight protocol was restricted to these 10 MPs reflecting both extensors and flexors, as illustrated in the MYOTONES body chart (Supplementary Fig. S5).Precise anatomical locations are described in detail elsewhere 40 .

Myoton technology and protocol
The principle of Myoton technology (Myoton AS, Estonia) 36 is based on natural damped oscillations of biological soft tissue.The Myoton device produces recordings of five parameters (stiffness, non-neural tone, elasticity, relaxation, creep) but stiffness was selected for a mechanical dynamic response and as digital biomarker for the present study, as it is a well-understood tissue property and Myoton recordings have been validated using a multilayered phantom tissue model 23 and in human muscle 37 .Inter and intra-rater reliability have also been established in healthy participants for all body measurement point (MP) sites relevant to the MYOTONES protocol 29,40,42 .We did not use electromyography (EMG) during the Myoton sessions to examine values at rest (silent EMG signal) 70 due to several operational constraints (acknowledged in study limitations in the discussion).
After positioning the Myoton device perpendicular to the skin, the rounded tip of the probe applies a brief (15 ms), low force (0.4 Newton [N]) mechanical impulse with a constant preload (0.18 N).The present study analyzed passive muscle stiffness (S) recorded by damped oscillations as numerical parameters [N/m] of superficial skeletal muscle, tendon and a fascia, which is a meaningful biomechanical characteristic currently best understood in relation to soft biological tissue structure and composition 74 .Stiffness (S) is a measure of a tissue's ability to resist an external force that modifies its shape and is calculated using the formula: S = a max • m probe /ΔI; a = acceleration, m = mass of measurement mechanism, ΔI = maximum displacement, reflected as newton-meter [N/m] calculated by internal device algorithm (MyotonPRO User Manual rev17, 14th Nov. 2017).For each MP, five impulses were applied and a mean value was calculated.Coefficient of variation (CV) for the five impulses was accepted if it was lower than 3% (%), which usually only required one measurement set of impulses but measurement was repeated if the CV was > 3%.Data sampling on the ground was performed in a dedicated quiet room with the participant completely relaxed, according to a standardized protocol (sitting or lying on guerney For baseline data collection (BDC) sessions the MyotonPRO was used.For inflight sessions, a space-qualified MyotonPRO device (OHB Space Systems Inc., Bremen, Germany) was used (with manual ON/OFF switch); launched by NASA contractor SpaceX (Falcon 9, CRS rocket) to the ISS.

Skin thermal imaging
Body and skin temperature are critical to a muscle's biomechanical properties 32 .To monitor skin temperature at MPs, a portable thermal camera imaging system (FLIR T640, RS Components GmbH, Möhrfelden-Walldorf, Germany) was used during pre-and postflight BDC sessions prior to Myoton measurements (Supplementary Table S1).Cabin temperature during inflight experimental sessions was recorded from the cabin log files inside the Columbus module (between range ~ 22-24 °C).

Ultrasound imaging
At the first BDC session, ultrasound imaging was used prior to Myoton measurement to verify anatomical location of MPs (Supplementary Figs.S2 and S3).In subsequent sessions, ultrasound followed Myoton data collection to document superficial tissue thickness (skin, subcutaneous fat, perimuscular fascia).Images were taken using a real-time B-mode ultrasound scanner (ORCHEO lite, SONOSCANNER, Paris or SUPERSONIC Aixplorer, SuperSonic Imagine, Aix-en-Provence) with a linear transducer (2-18 MHz).Inflight ultrasound sessions were performed by crew operators with a scientific team member (P.E.M., M.J.S. or M.B.W.) guiding remotely from the ground facility (CADMOS, Toulouse, France).All images were measured later off-line by one investigator (P.E.M.) using custom written Matlab (Mathworks, USA) code (written by M.B.W.).Ultrasound imaging of musculoskeletal soft tissues is a well-established valid and reliable tool, and reliability of the present protocol was demonstrated 40 .

Baseline data collection (BDC): pre-and postflight
Anatomical location of all 10 MPs was conducted by manual palpation using easily palpable bone landmarks (e.g.acromion, vertebral spinous processes [C7, L4]), following known clinical protocols 40 .The MPs were marked on the skin using a surgical skin marker pen (Dermaskript, pmfmedical.com).Custom-made adhesive rulers (cm) were used for accurate relocation of MPs, followed by photo documentation of individual MPs to create an illustrated personalized manual as a reference for Crew members for subsequent sessions on the ISS.The participant was fully relaxed for all measurements and the first set for MP1-5 were performed in prone lying, with the hands and forearms relaxed by the sides of the body and a small pillow placed under the ankles.MP6 was measured in upright sitting, arms resting on the thighs.The participant then moved into a supine position, with a pillow under the knees, for measuring MP7-10.All MPs were only studied on the right side of the body, regardless of dominance, to minimize errors in the protocol, as it was critical to avoid wasted data.During all pre-and postflight BDC sessions, thermal FLIR camera imaging system was used to monitor skin temperature.Measurements with the MyotonPRO were then taken of MPs followed by ultrasound imaging.Before launch, astronauts received a 1 h on-the-ground training on locating anatomical structures and MPs, and using spacequalified Myoton technology and the ultrasound imaging device.Most participants were also operators inflight, so were trained.If a crew member was only a participant, they were not trained.

Inflight data collection
According to ISS flight rules, countermeasure exercises including aerobic and resistance training were performed for up to 2-2.5 h/day for 6/week by all astronauts whilst inflight 7,45 .
Inflight Myoton data collection was performed on four different flight days (FD5-15, FD31-60, FD121-150, Return minus [R-]10 ± 5, i.e. before return to Earth) prior to daily activities (e.g.maintenance activities, onboard exercise protocols).Ultrasound imaging was performed on two flight days (FD31-60, R-10 ± 5).Data were collected by a fellow crew member (operator) using the illustrated personalized manual (Crew iPad) for reference.The participant was fully relaxed in prone (MPs 1-6) and supine (MPs 7-10) positions, with the body loosely fixed to the cabin floor with a pelvic belt to minimize movements (free-floating body shifts) to allow for passive muscle stiffness measurements.The operator used feet hooks and floor bars for their own body stabilization to enable accurate data collection from each of the skin MPs (see Supplementary Fig. S5).

Statistical analysis
GraphPad Prism (GraphPad Prism 9.5.0 for Windows, GraphPad Software, San Diego, California, USA, www.graph pad.com) was used to process all data.Myoton data are intentionally provided as absolute values (colourcoded data clusters for each study participant for three conditions, pre/in/postflight) in scatter plots for data interpretation (at a glance) necessary for the comparison between muscle adaptation/maladaptation effects in healthy Astronauts (thus lacking pathologies or diagnosed diseases/co-morbidities usually investigated in clinical studies).Absolute values enable evaluation of the data with respect to the standard error of measurement, providing confidence in the robustness of the data, and changes in values greater than the MDC (whether increased or decreased from the mean) indicate abnormal stiffness.The Shapiro-Wilk test examined for normality of data distribution and showed a parametric distribution.Thus, one-way ANOVA with repeated measures was used, which if significant was followed by Tukey's post hoc test, to compare passive muscle stiffness given as Newtons per metre [N/m] at different time points (preflight, inflight, postflight).Data [N/m] are presented as mean and standard deviation (SD) in 2D scatter/dot plots using participant-matched colour-coding points (Astronauts A to S) plotted against pre/in/postflight condition (circle/square/triangle data point symbols) for visualization www.nature.com/scientificreports/(Figs. 1 and 2, Supplementary Fig. S1).The level of significance was set at p = 0.05 (pre/in/postflight).Changes in stiffness inflight and postflight were examined with separate linear mixed models using SPSS.(Fig. 3).Visualization of the case-wise relationship between number of days and stiffness suggested a quadratic relationship for both inflight and postflight.The quadratic was then calculated for number of days for each participant for both inflight and postflight days.The days were centered with respect to the first day inflight or first day postflight.Multiple models were developed where combinations of fixed and random effects consisting of intercept, number of days and quadratic days were used as covariates to predict the dependent variable of stiffness for each MP.The quality of fit for each model was assessed using the Akaike Information Criterion.For all measurement sites, both inflight and postflight, the best fitting model consisted of fixed effects of intercept and quadratic days, with random effect of intercept.If not indicated otherwise, MDC reference values from the 10 skin MPs were used 40 , which were produced from reliability studies involving a minimum of 20 participants.

Ethical approval
This